This invention relates to the use of compounds as inhibitors of the fatty acid synthase FabH.
The pathway for the biosynthesis of saturated fatty acids is very similar in prokaryotes and eukaryotes. However, although the chemical reactions may not vary, the organization of the biosynthetic apparatus is very different. Vertebrates and yeasts possess type I fatty acid synthases (FASs) in which all of the enzymatic activities are encoded on one or two polypeptide chains, respectively. The acyl carrier protein (ACP) is an integral part of the complex. In contrast, in most bacterial and plant FASs (type II) each of the reactions are catalyzed by distinct monofunctional enzymes and the ACP is a discrete protein. Mycobacteria are unique in that they possess both type I and II FASs; the former is involved in basic fatty acid biosynthesis whereas the latter is involved in synthesis of complex cell envelope lipids such as mycolic acids. There therefore appears to be considerable potential for selective inhibition of the bacterial systems by broad-spectrum antibacterial agents (Jackowski, S. 1992. In Emerging Targets in Antibacterial and Antifungal Chemotherapy. Ed. J. Sutcliffe and N. Georgopapadakou. Chapman and Hall, New York; Jackowski, S. et al. (1989). J. Biol. Chem. 264, 7624-7629.)
The first step in the biosynthetic cycle is the condensation of malonyl-ACP with acetyl-CoA by FabH. In subsequent rounds malonyl-ACP is condensed with the growing-chain acyl-ACP (FabB and FabF, synthases I and II respectively). The second step in the elongation cycle is ketoester reduction by NADPH-dependent xcex2-ketoacyl-ACP reductase (FabG). Subsequent dehydration by xcex2-hydroxyacyl-ACP dehydrase (either FabA or FabZ) leads to trans-2-enoyl-ACP which is in turn converted to acyl-ACP by NADH-dependent enoyl-ACP reductase (FabI). Further rounds of this cycle, adding two carbon atoms per cycle, eventually lead to palmitoyl-ACP whereupon the cycle is stopped largely due to feedback inhibition of FabH and I by palmitoyl-ACP (Heath, et al, (1996), J.Biol.Chem. 271, 1833-1836). FabH is therefore a major biosynthetic enzyme which is also a key regulatory point in the overall synthetic pathway (Heath, R. J. and Rock, C. O. 1996. J.Biol.Chem. 271, 1833-1836; Heath, R. J. and Rock, C. O. 1996. J.Biol.Chem. 271, 10996-11000).
The antibiotic thiolactomycin has broad-spectrum antibacterial activity both in vivo and in vitro and has been shown to specifically inhibit all three condensing enzymes. It is non-toxic and does not inhibit mammalian FASs (Hayashi, T. et al., 1984. J. Antibiotics 37, 1456-1461; Miyakawa, S. et al., 1982. J. Antibiotics 35, 411-419; Nawata, Y et al., 1989. Acta Cryst. C45, 978-979; Noto, T. et al., 1982. J. Antibiotics 35, 401-410; Oishi, H. et al., 1982. J. Antibiotics 35, 391-396. Similarly, cerulenin is a potent inhibitor of FabB and F and is bactericidal but is toxic to eukaryotes because it competes for the fatty-acyl binding site common to both FAS types (D""Agnolo, G. et al.,1973. Biochim. Biophys. Acta. 326, 155-166). Extensive work with these inhibitors has proved that these enzymes are essential for viability. Little work has been carried out in Gram-positive bacteria.
There is an unmet need for developing new classes of antibiotic compounds that are not subject to existing resistance mechanisms. No marketed antibiotics are targeted against fatty acid biosynthesis, therefore it is unlikely that novel antibiotics of this type would be rendered inactive by known antibiotic resistance mechanisms. Moreover, this is a potentially broad-spectrum target. Therefore, FabH inhibitors would serve to meet this unmet need.
This invention comprises indole derivatives and pharmaceutical compositions containing these compounds and their use as FabH inhibitors that are useful as antibiotics for the treatment of Gram positive and Gram negative bacterial infections.
This invention further constitutes a method for treatment of a Gram negative or Gram positive bacterial infection in an animal, including humans, which comprises administering to an animal in need thereof, an effective amount of a compound of this invention.
The compounds of this invention are represented by Formula (I): 
wherein,
R1 is selected from the group consisting of H, CO2R4, COR4, CONR5R6, CH(OH)R4, CR4xe2x95x90NOR4, heteroaryl and substituted heteroaryl;
R2 is selected from the group consisting of H, COR4, and CH(OH)R4;
R3 is selected from the group consisting of aryl, substituted aryl, heteroaryl and substituted heteroaryl;
R4 is H or lower alkyl;
R5 and R6 are, independently, H, or lower alkyl or, together, form a 5 or 6 membered ring selected from the group consisting of piperidine, piperazine, pyrrolidine, morpholine and hydroxy piperidine; and n is an integer from 1 to 6.
Also included in the invention are pharmaceutically acceptable salt complexes.
Preferred substituted heteroaryl moieties include oxadiazole and oxazole.
As used herein, xe2x80x9calkylxe2x80x9d means both straight and branched chains of 1 to 10 carbon atoms, unless the chain length is otherwise limited, including, but not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl and the like. The alkyl may carry substituents such as hydroxy, carboxy, alkoxy, and the like.
The term xe2x80x9ccycloalkylxe2x80x9d is used herein to mean cyclic rings, preferably of 3 to 8 carbons, including but not limited to cyclopropyl, cyclopentyl, cyclohexyl, and the like.
The term xe2x80x9carylalkylxe2x80x9d or xe2x80x9cheteroarylalkylxe2x80x9d or xe2x80x9cheterocyclicalkylxe2x80x9d is used herein to mean C1-10 alkyl, as defined above, attached to an aryl, heteroaryl or heterocyclic moiety, as also defined herein, unless otherwise indicated.
As used herein, xe2x80x9carylxe2x80x9d means phenyl and naphthyl and substituted aryl such as hydroxy, carboxy, halo, alkoxy, methylenedioxy, and the like.
As used herein, xe2x80x9cheteroarylxe2x80x9d means a 5-10 membered aromatic ring system in which one or more rings contain one or more heteroatoms selected from the group consisting of N, O or S, such as, but not limited, to pyrrole, pyrazole, furan, thiophene, quinoline, isoquinoline, quinazolinyl, pyridine, pyrimidine, oxazole, thiazole, thiadiazole, triazole, imidazole, and benzimidazole.
As used herein, preferred aryl substituents include halo, including chloro, fluoro, bromo and iodo, in any combination; C1-10 alkyl, C1-10 alkoxy, aryloxy, or heteroaryloxy.
The compounds of this invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active forms. All of these compounds and diastereomers are contemplated to be within the scope of the present invention.
Some of the compounds of this invention may be crystallised or recrystallised from solvents such as organic solvents. In such cases solvates may be formed. This invention includes within its scope stoichiometric solvates including hydrates as well as compounds containing variable amounts of water that may be produced by processes such as lyophilisation.
Since the antibiotic compounds of the invention are intended for use in pharmaceutical compositions it will readily be understood that they are each provided in substantially pure form, for example at least 60% pure, more suitably at least 75% pure and preferably at least 85%, especially at least 95% pure, particularly at least 98% pure (% are on a weight for weight basis). Impure preparations of the compounds may be used for preparing the more pure forms used in the pharmaceutical compositions; these less pure preparations of the compounds should contain at least 1%, more suitably at least 5% and preferably from 10 to 49% of a compound of the formula (I) or salt thereof.
Preferred compounds of the present invention include:
5-(2,6-dichlorobenzyloxy)-1-[3-(1H-tetrazol-5-yl)propyl]-1H-indole-2-carboxylic acid ethyl ester;
1-{5-(2,6-dichlorobenzyloxy)-1-[3-(1H-tetrazol-5-yl)propyl]-1H-indol-2-yl}-1-morpholin-4-yl-methanone;
5-(2,6-dichlorobenzyloxy)-1-[3-(1H-tetrazol-5-yl)propyl]-1H-indole-2-carboxylic acid isobutyl amide;
5-(2,6-dichlorobenzyloxy)-1-[3-(1H-tetrazol-5-yl)propyl]-1H-indole-2-carboxylic acid diethylamide;
5-(2,6-dichlorobenzyloxy)-3-formyl-1-[3-(1H-tetrazol-5-yl)propyl]-1H-indole-2-carboxylic acid ethyl ester;
5-(2,6-dichlorobenzyloxy)-2-(3-methyl-[1,2,4]oxadiazol-5-yl)-1-[3-(1H-tetrazol-5-yl)propyl]-1H-indole;
5-(2,6-dichlorobenzyloxy)-1-[3-(1H-tetrazol-5-yl)propyl]-1H-indole;
1-{5-(2,6-dichlorobenzyloxy)-1-[3-(1H-tetrazol-5-yl)-propyl]-1H-indol-3-yl}propan-1-one;
5-(2,6-dichlorobenzyloxy)-1-[3-(1H-tetrazol-5-yl)propyl]-1H-indole-2-carbaldehyde-O-methyl oxime; and
5-(2,6-dichlorobenzyloxy)-2-(oxazol-5-yl)-1-[3-(1H-tetrazol-5-yl)propyl]-1H-indole.
Compounds of formula (I) wherein R1 is an ethyl ester, R2 is H, R3 is 2,6-dichlorophenyl and n=3 were prepared by the method described in Scheme 1. 
a) 10% Pd/C, H2, EtOH; b) 2,6-dichiorobenzyl chloride, Cs2CO3, DMF; c) NaH, 4-bromobutyronitrile, DMF; d) Me3Si-N3, Bu2Snxe2x95x90O, toluene, reflux
Indole ethyl ester 1, Scheme 1, (Aldrich) was debenzylated via catalytic hydrogenation to provide the 5-hydroxy indole 2. The 5-hydroxyl was then alkylated using the desired halide-containing reagent, in this example 2,6-dichlorobenzyl chloride, using a suitable base such as cesium or potassium carbonate, providing indole 3. The indole nitrogen was next alkylated with 4-bromobutyronitrile, using NaH as a base, to provide the tetrazole precursor 4. The nitrile moiety of 4 was next converted to the tetrazole via reaction with azide according to published methods. In this particular example, the method using azidotrimethylsilane and dibutyltin oxide in refluxing toluene was chosen. Thus tetrazole 5 was obtained.
Compounds of formula (I) wherein R1 is an amide, morpholino amide for this example, R2 is H, R3 is 2,6-dichlorophenyl and n=C3 were prepared by the method described in Scheme 2. 
a) 1N NaOH, THF, MeOH; b) PyBrop, morpholine, Konig""s base, DMAP, DMF; c) Me3Si-N3, Bu2Snxe2x95x90O, toluene, reflux
Indole ethyl ester 4, Scheme 2, was saponified to the corresponding carboxylic acid 6 using aqueous NaOH, THF and MeOH. The acid was then converted to an amide using standard amide coupling reagents and the desired amine. In this example, the amine used was morpholine and the coupling reagent was PyBrop in the presence of Konig""s base (diisopropylethylamine) and catalytic DMAP. This provided morpholino amide 7. Nitrile 7 was then converted to the tetrazole 8 according to the method outlined in Scheme 1.
Compounds of formula (I) wherein R1 is an ethyl ester, R2 is CHO, R3 is 2,6-dichlorophenyl and n=3 were prepared by the method described in Scheme 3. 
a) POCl3, DMF; b) Me3Si-N3, Bu2Snxe2x95x90O, toluene, reflux
Indole ethyl ester 4, Scheme 3, was converted to the C-3 aldehyde 9 using a Vilsmeier reaction (POCl3, DMF). Nitrile 9 was then converted to the tetrazole 10 according to the method outlined in Scheme 1.
Compounds of formula (I) wherein R1 is H, R2 is H, R3 is 2,6-dichlorophenyl and n=3 were prepared by the method described in Scheme 4. 
a) 2,6-dichlorobenzyl bromide, Cs2CO3, DMF; b) NaH, 4-bromobutyronitrile, DMF; c) NaOMe, MeOH; d) Me3Si-N3, Bu2Snxe2x95x90O, toluene, reflux
5-Hydroxyindole (Aldrich), Scheme 4, was converted to the 2,6-dichlorobenzylether 12 according to methods outlined in Scheme 1. The indole nitrogen was then alkylated with 4-bromobutyronitrile as previously described (Scheme 1). However, in this instance, a mixture of products was obtained consisting of the desired nitrile 13 and the undesired product 14 in a ratio of 85:15. The undesired nitrile (14) was selectively decomposed by treating the mixture directly with sodium methoxide in MeOH providing 13 as the sole product. Nitrile 13 was then converted to the tetrazole 15 as previously outlined.
Compounds of formula (I) wherein R1 is H, R2 is ethyl ketone (xe2x80x94COEt), R3 is 2,6-dichlorophenyl and n=3 were prepared by the method described in Scheme 5. 
a) 2,6-dichlorobenzyl bromide, Cs2CO3, DMF; b) NaH, 4-bromobutyronitrile, DMF; c) i. POCl3, DMF, ii. separate 16 from 17; d) EtMgBr, THF; e) Me3Si-N3, Bu2Snxe2x95x90O, toluene, reflux
The mixture of nitriles, 13 and 14, were prepared as outlined in Scheme 4, however, in this instance the two compounds were not separated. The mixture was subjected to the Vilsmeier reaction (POCl3, DMF) to provide the mixture of aldehydes, 16 and 17, which were readily separated using flash column chromatography. The pure aldehyde 16 was then reacted with the commercially available Grignard reagent ethyl magnesium bromide in an aprotic solvent such as THF or ether. Any desired Grignard or organo lithium reagent can be used at this stage. While the normal product from the Grignard reaction would be the secondary alcohol, in this example, the ketone 18 was isolated directly from the reaction mixture in low yield. The nitrile group of 18 was then converted to the desired tetrazole as previously described, thus providing 19.
Compounds of formula (I) wherein R1 is a substituted oxadiazole, R2 is H, R3 is 2,6-dichlorophenyl and n=3 were prepared by the method described in Scheme 6. 
a) 1N NaOH, THF, EtOH; b) i. CDI, DMF, ii. NH3, DMF; c) Me2NCMe(OMe)2, 110xc2x0 C.; d) NH2OH-HCl, AcOH, dioxane, 1N NaOH, 90xc2x0 C.; e) Me3Si-N3, Bu2Snxe2x95x90O, toluene, reflux
Nitrilexe2x80x94ester intermediate 4 was hydrolyzed to the corresponding carboxylic acid 20. Acid 20 was then activated using carbonyl diimidazole (CDI) followed by treatment with gaseous ammonia to form the primary amide 21. Reaction of 21 with dimethylacetamide dimethylacetal provided 22 which was then converted into the desired oxadiazole system via treatment with hydroxylamine, thus providing 23. The nitrile functional group of 23 was next converted to the tetrazole 24 by reaction with azide according the previously described methods.
Compounds of formula (I) wherein R1 is an oxazole, R2 is H, R3 is 2,6-dichlorophenyl and n=3 were prepared by the method described in Scheme 7. 
a) LiBH4, THF; b)MnO2, CH2Cl2; c) tol-SO2CH2NC, MeOH; d) Me3Si-N3, Bu2Snxe2x95x90O, toluene, reflux
The ethyl ester of 4 was reduced to the corresponding primary alcohol 25 using lithium borohydride in THF. Oxidation with MnO2 provided the aldehyde 26. Alternative oxidizing reagents such as Dess-Martin reagent, Swern oxidation or pyridine-SO3 could also be used. Reaction of the aldehyde with tosylmethyl isocyanide yielded the oxazole 27 which was next converted to tetrazole 28 following standard methods.
Compounds of formula (I) wherein R1 is an O-methyl oxime, R2 is H, R3 is 2,6-dichlorophenyl and n=3 were prepared by the method described in Scheme 8. 
a) NH2OMe.HCl, b) Me3Si-N3, Bu2Snxe2x95x90O, toluene, reflux
Aldehyde 26 was converted to the desired O-methyl oxime 29 via reaction with methoxyamine. The nitrile was then converted to the tetrazole 30 by reaction with azide as previously described.
Any of these compounds can potentially be used to treat any disease caused by pathogens that possess a type II fatty acid synthesis pathway, such as mycobacteria. Such diseases include, but are not limited to, malaria and tuberculosis.